Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapy for patients with leukemia or lymphoma. However, chronic graft versus host-disease (cGVHD) remains a significant cause of late morbidity and mortality (Socie G (2011) Chronic GVHD: a new risk score? Blood 117:6408-6409; Socie G, et al. (2011) Chronic graft-versus-host disease: long-term results from a randomized trial on graftversus-host disease prophylaxis with or without anti-T-cell globulin ATG-Fresenius. Blood 117:6375-6382; Kohrt H E, et al. (2009) TLI and ATG conditioning with low risk of graft-versus-host disease retains antitumor reactions after allogeneic hematopoietic cell transplantation from related and unrelated donors. Blood 114:1099-1109; Arai S, et al. (2012) Prophylactic rituximab after allogeneic transplantation decreases B-cell alloimmunity with low chronic GVHD incidence. Blood 119:6145-6154).
Several studies indicate that donor-derived alloreactive B and T cells are involved in pathogenesis of cGVHD. In support of a B cell role (Arai S, et al. (2012) Prophylactic rituximab after allogeneic transplantation decreases B-cell alloimmunity with low chronic GVHD incidence. Blood 119:6145-6154; Allen J L, et al. (2012) B cells from patients with chronic GVHD are activated and primed for survival via BAFF-mediated pathways. Blood 120:2529-2536; Kuzmina Z, et al. (2011) Significant differences in B-cell subpopulations characterize patients with chronic graft-versus-host disease-associated dysgammaglobulinemia. Blood 117:2265-2274; Sarantopoulos S, et al. (2007) High levels of B-cell activating factor in patients with active chronic graftversus-host disease. Clin Cancer Res 13:6107-6114; Sarantopoulos S, et al. (2011) Recovery of B-cell homeostasis after rituximab in chronic graft-versus-host disease. Blood 117:2275-2283; She K, et al. (2007) Altered Toll-like receptor 9 responses in circulating B cells at the onset of extensive chronic graft-versus-host disease. Biol Blood Marrow Transplant 13:386-397), the presence of circulating autoantibody (Sarantopoulos S, et al. (2009) Altered B-cell homeostasis and excess BAFF in human chronic graft-versushost disease. Blood 113:3865-74) and alloantibody (Miklos D B, et al. Antibody response to DBY minor histocompatibility antigen is induced after allogeneic stem cell transplantation and in healthy female donors. Blood 103:353-359; Wechalekar A, Cranfield T, Sinclair D, and Ganzckowski M (2005) Occurrence of autoantibodies in chronic graft vs. host disease after allogeneic stem cell transplantation. Clin Lab Haematol 27:247-249) have been associated with development of cGVHD. Specifically, predominant B cell subsets have been demonstrated in patients with cGVHD and identified in different studies as naïve (Kuzmina Z, et al. (2011) Significant differences in B-cell subpopulations characterize patients with chronic graft-versus-host disease-associated dysgammaglobulinemia. Blood 117:2265-2274) and post germinal center B cells (Sarantopoulos S, et al. (2007) High levels of B-cell activating factor in patients with active chronic graftversus-host disease. Clin Cancer Res 13:6107-6114; Sarantopoulos S, et al. (2011) Recovery of B-cell homeostasis after rituximab in chronic graft-versus-host disease. Blood 117:2275-2283; Sarantopoulos S, et al. (2009) Altered B-cell homeostasis and excess BAFF in human chronic graft-versushost disease. Blood 113:3865-74). In addition, B cell related markers and antibodies have been recognized as biomarkers for characterization and scoring cGVHD (Schultz K R, et al. (2006) Toward biomarkers for chronic graft-versus-host disease: National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: III. Biomarker Working Group Report. Biol Blood Marrow Transplant 12:126-137; Socie G (2011) Chronic GVHD: B cells come of age. Blood 117:2086-2087). Finally, Rituximab, which depletes B cells, has been successfully used as cGVHD therapy (Sarantopoulos S, et al. (2011) Recovery of B-cell homeostasis after rituximab in chronic graft-versus-host disease. Blood 117:2275-2283; Deneberg S, Lerner R, Ljungman P, Ringden O, and Hagglund H (2007) Relapse of preB-ALL after rituximab treatment for chronic graft versus host disease: implications for its use? Med Oncol 24:354-356; Kharfan-Dabaja, M A and Bazarbachi A (2010) Emerging role of CD20 blockade in allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 16:1347-1354; Kharfan-Dabaja M A, et al. (2009) Efficacy of rituximab in the setting of steroid-refractory chronic graftversus-host disease: a systematic review and meta-analysis. Biol Blood Marrow Transplant 15:1005-1013; Zaja, F, et al. (2007) Treatment of refractory chronic GVHD with rituximab: a GITMO study. Bone Marrow Transplant 40:273-277; Cutler C, et al. (2006) Rituximab for steroid-refractory chronic graft-versus-host disease. Blood 108:756262).
Previous studies by our group have shown alloantibody responses occur in male HCT patients with female donors (F→M). These responses include donor derived alloreactive IgG that recognizes one or more Y chromosome encoded proteins (H-Y antigens), including the DDX3Y protein (referred to hereafter as DBY) and its immunodominant DBY-2 peptide, which we use in studies here. In addition, donor-derived anti-DBY antibodies appear in serum in association with cGVHD in F→M patients implicating alloreactive B cells in cGVHD pathogenesis (Miklos D B, et al. Antibody response to DBY minor histocompatibility antigen is induced after allogeneic stem cell transplantation and in healthy female donors. Blood 103:353-359; Miklos D B, et al. (2005) Antibody responses to H-Y minor histocompatibility antigens correlate with chronic graft-versus-host disease and disease remission. Blood 105:2973-2978). In order to test the hypothesis that H-Y specific B cells contribute to cGVHD pathogenesis, we have developed an H-Y specific FACS stain for their isolation and characterization.
Here, we demonstrate that 6 months after F→M transplant, more than half of 28 male patients with female donors develop circulating B cells whose surface IgM and IgG receptors specifically bind DBY-2, and hence are poised to undergo class switch and differentiate to plasma cells that produce IgG anti-DBY-2 antibodies. Further, we show that their presence in circulation is strongly associated with the development of cGVHD (p=0.004), that is, the overwhelming majority (15/16) of patients who have DBY-2 specific B cells either have or will develop cGVHD within 1-3 months. In contrast, only about half (5/12) of patients who do not have these B cells develop cGVHD. We detected immunoglobulin (Ig) M and IgG anti-DBY-2 B cells in all but 2 of the patients who later developed circulating IgG anti-DBY-2 (p=0.002).
As is usual in studies with antigen binding B cells in the mouse (Yang Y, et al. (2012) Antigen-specific memory in B-1a and its relationship to natural immunity. Proc Natl Acad Sci USA 109:5388-5393; Yang Y, et al. (2012) Antigen-specific antibody responses in B-1a and their relationship to natural immunity. Proc Natl Acad Sci USA 109:5382-5387), the amount of the antigen bound to the B cells is strongly correlated with the amount of surface Ig on the cells, which at the time point we examined is exclusively IgM and IgD associated mainly with Igλ light chains. However, even though these cells have most likely arisen in response to antigenic stimulation (DBY-2 on the male patient's cells stimulating female donor B cells), they express a phenotype (CD19+IgM+IgD+CD38+ and CD27−) commonly taken as characteristic of transitional B cells that have recently entered circulation from bone marrow.
The prospective monitoring of anti-DBY-2 B cells may direct a more effective schedule for alloreactive B cell depletion therapy towards a goal of cGVHD prevention. Likewise, DBY-2 B cell monitoring may help elucidate whether current in vivo B cell depletion therapy for cGVHD effectively depletes these alloreactive B cells or if they persist and proliferate when cGVHD recurs.